Biology subjects

Merrikh, H.

Publications and source records attributed to Merrikh, H..

15 recordsLinked to original sources

bioRxiv2026

Polynucleotide Phosphorylase Stops Replication Restart by Degrading the RNA within R-loops during Replication-Transcription Conflicts

DNA replication and transcription machineries function simultaneously, on the same template, leading to conflicts between the two. During conflicts, the nascent mRNAs can hybridize to their template strands, generating RNA:DNA hybrids, which reside within nucleic acid structures known as R-loops. Accumulation of R-loops can lead to severe replication fork stalling, eventually leading to cell death. In bacteria, there are well-known replication restart mechanisms that should rescue these stalled replication forks, and yet, during conflicts, this process is inhibited. Additionally, in vitro work has shown that RNA:DNA hybrids are substrates for replication restart. Here, we discovered that the highly conserved exonuclease Polynucleotide Phosphorylase (PNPase in Bacillus subtilis) prevents replication restart from RNA:DNA hybrids, specifically at regions of severe conflicts where R-loops accumulate. Our in vitro data show that PNPase binds RNA:DNA hybrids and digests the RNA in these structures. Consistently, we found that in vivo, PNPase binds to conflict regions and reduces R-loop levels. To our knowledge, the only other class of enzymes known to digest the RNA from hybrids are RNases H. Our findings identify PNPase as a new enzyme that can perform a similar function. Importantly, our data show that PNPase inhibits replication restart from R-loops and its absence allows for replication restart from conflict regions. We also observed that PNPase activity reduces mutations, suggesting that replication restart from RNA:DNA hybrids is highly mutagenic. We propose that PNPase acts as a safeguard against mutagenic replication restart from RNA:DNA hybrids within R-loops by digesting the RNA which could re-initiate replication.

genetics↗

bioRxiv2026

Processing of Reversed Replication Forks is Required for the Resolution of Replication-Transcription Conflicts

DNA replication and transcription occur simultaneously on the same template, leading to conflicts between the two machineries. Conflicts stall replication forks, lead to genome instability, mutagenesis, and the formation of deleterious R-loop structures. There are two types of conflicts: depending on the strand where a gene is encoded, the two machineries either meet head-on or co-directionally. The adverse outcomes of conflicts in the head-on orientation are significantly more detrimental to cells compared to co-directional conflicts. Despite many studies across various organisms, how the replication fork structure is impacted by these encounters remains unclear. Here, we performed an unbiased genetic screen using a transposon library to identify factors essential for surviving head-on conflicts in Bacillus subtilis. Our screen identified three hits: RNase HIII, AddA and AddB. Our prior work had shown that RNase HIII, which processes R-loops, is essential for surviving head-on conflicts. However, AddA and AddB, which function together as a complex to process blunt DNA ends, have not been previously identified as essential conflict resolution factors. Through follow-up genetic and biochemical analyses, we found that the helicase activity but not the nuclease activity of this complex is required for conflict resolution. Based on the fundamental properties of DNA at replication fork structures, our work collectively indicates that upon head-on conflicts, the nascent strands form a reversed fork structure, which is then unwound by AddAB, which leads to re-annealing to the parental strands. This process re-establishes an intact replication fork that can be used to restart replication after conflicts with transcription.

molecular biology↗

bioRxiv2026

A universal regulatory mechanism for prevention of replication restart from RNA:DNA hybrids

RNA:DNA hybrids form across all genomes. In vitro studies showed that these structures can be used to restart stalled replication forks, especially upon replication-transcription conflicts, however, this has not been tested in vivo. Here, we identify a mechanism that prevents replication restart from hybrids within cells. We identified the tRNA ligase, AsnRS, as a regulator of replication restart from hybrids at conflict regions in Bacillus subtilis. We determined that the DNA translocase Mfd and DNA polymerase PolA play key roles in this pathway. We unraveled a mechanism whereby Mfd removes RNA Polymerases, exposing 3 ends of the RNAs within hybrids. These 3 ends are then capped by AsnRS, preventing restart by PolA. Remarkably, the mammalian homologs of AsnRS, NARS1 and NARS2, fully complement the AsnRS phenotypes in bacteria. We propose that this is a universally conserved mechanism that prevents untimely replication initiation outside of origins from bacteria to humans.

genetics↗

bioRxiv2023

Pathogenic bacteria experience pervasive RNA polymerase backtracking during infection

Pathogenic bacteria and their eukaryotic hosts are in a constant arms race. Hosts have numerous defense mechanisms at their disposal that not only challenge the bacterial invaders, but have the potential to disrupt molecular transactions along the bacterial chromosome. However, it is unclear how the host impacts association of proteins with the bacterial chromosome at the molecular level during infection. This is partially due to the lack of a method that could detect these events in pathogens while they are within host cells. We developed and optimized a system capable of mapping and measuring levels of bacterial proteins associated with the chromosome while they are actively infecting the host (referred to as PIC-seq). Here, we focused on the dynamics of RNA polymerase (RNAP) movement and association with the chromosome in the pathogenic bacterium Salmonella enterica as a model system during infection. Using PIC-seq, we found that RNAP association patterns with the chromosome change during infection genome-wide, including at regions that encode for key virulence genes. Importantly, we found that infection of a host significantly increases RNAP backtracking on the bacterial chromosome. RNAP backtracking is the most common form of disruption to RNAP progress on the chromosome. Interestingly, we found that the resolution of backtracked RNAPs via the anti-backtracking factors GreA and GreB is critical for pathogenesis, revealing a new class of virulence genes. Altogether, our results strongly suggest that infection of a host significantly impacts transcription by disrupting RNAP movement on the chromosome within the bacterial pathogen. The increased backtracking events have important implications not only for efficient transcription, but also for mutation rates as stalled RNAPs increase the levels of mutagenesis.

biochemistry↗

bioRxiv2022

A small molecule that inhibits the evolution of antibiotic resistance

Antimicrobial resistance (AMR) rapidly develops against almost all available therapeutics. New antibiotics target essential processes in bacteria but fail to address the root of the problem: mutagenesis and evolution. We recently proposed that inhibiting the molecular mechanisms underlying bacterial evolution is the ultimate solution to preventing AMR development. Here, we describe the first compound that inhibits the occurrence and progression of AMR by directly targeting a highly conserved bacterial evolvability factor, Mfd. We previously found that this RNA polymerase-associated translocase is required for rapid AMR development across highly divergent pathogens. Through an in vivo screen, we identified 43 potential Mfd-inhibiting compounds. Here we present on target validation, biochemical characterization, and in vivo efficacy studies of a lead compound, referred to as ARM-1. ARM-1 binds Mfd and modulates its RNA polymerase interaction. Inhibition of Mfd activity by ARM-1 delays the development of mutations and resistance acquisition, both in pure culture and during infection. Importantly, our data show that this compound prevents the evolution of AMR across highly divergent pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium. The novel compound we present here has the potential to develop into a clinically useful "anti-evolution" drug. This work demonstrates that the molecular mechanisms of evolution are pharmaceutically targetable, and that this strategy could help prevent AMR development.

microbiology↗

bioRxiv2022

The high-resolution in vivo measurement of replication fork velocity and pausing

An important step towards understanding the mechanistic basis of the central dogma is the quantitative characterization of the dynamics of nucleic-acid-bound molecular motors in the context of the living cell, where a crowded cytoplasm as well as competing and potentially antagonistic processes may significantly affect their rapidity and reliability. To capture these dynamics, we develop a novel method, lag-time analysis, for measuring in vivo dynamics. The approach uses exponential growth as the stopwatch to resolve dynamics in an asynchronous culture and therefore circumvents the difficulties and potential artifacts associated with synchronization or fluorescent labeling. Although lag-time analysis has the potential to be widely applicable to the quantitative analysis of in vivo dynamics, we focus on an important application: characterizing replication dynamics. To benchmark the approach, we analyze replication dynamics in three different species and a collection of mutants. We provide the first quantitative locus-specific measurements of fork velocity, in units of kb per second, as well as replisome-pause durations, some with the precision of seconds. The measured fork velocity is observed to be both locus and time dependent, even in wild-type cells. In addition to quantitatively characterizing known phenomena, we detect brief, locus-specific pauses at rDNA in wild-type cells for the first time. We also observe temporal fork velocity oscillations in three highly-divergent bacterial species. Lag-time analysis not only has great potential to offer new insights into replication, as demonstrated in the paper, but also has potential to provide quantitative insights into other important processes.

biophysics↗

bioRxiv2022

Nucleotide excision repair is universally mutagenic and transcription-associated

In bacteria, mutations lead to the evolution of antibiotic resistance, which is one the main public health problems of the 21st century. Therefore, determining which cellular processes most frequently contribute to mutagenesis, especially in cells that have not been exposed to exogenous DNA damage, is critical. Here, we show that endogenous oxidative stress is a key driver of mutagenesis and the subsequent development of antibiotic resistance. This is the case for all classes of antibiotics tested and across highly divergent species, including patient-derived strains. We show that the transcription-coupled repair pathway, which uses the nucleotide excision repair proteins (TC-NER), is responsible for endogenous oxidative stress-dependent mutagenesis and subsequent evolution. This strongly suggests that a majority of mutations arise through transcription-associated processes rather than the replication fork. In addition to determining that the NER proteins play a critical role in mutagenesis and evolution, we also identify the DNA polymerases responsible for this process. Our data strongly suggest that cooperation between three different mutagenic DNA polymerases, likely at the last step of TC-NER, is responsible for mutagenesis and evolution. Overall, our work identifies that a highly conserved pathway drives mutagenesis due to endogenous oxidative stress, which has broad implications for all diseases of evolution, including antibiotic resistance development.

molecular biology↗

bioRxiv2021

Characterizing stochastic cell cycle dynamics in exponential growth

Two powerful and complementary experimental approaches are commonly used to study the cell cycle and cell biology: One class of experiments characterizes the statistics (or demographics) of an unsynchronized exponentially-growing population, while the other captures cell cycle dynamics, either by time-lapse imaging of full cell cycles or in bulk experiments on synchronized populations. In this paper, we study the subtle relationship between observations in these two distinct experimental approaches. We begin with an existing model: a single-cell deterministic description of cell cycle dynamics where cell states (i.e. periods or phases) have precise lifetimes. We then generalize this description to a stochastic model in which the states have stochastic lifetimes, as described by arbitrary probability distribution functions. Our analyses of the demographics of an exponential culture reveal a simple and exact correspondence between the deterministic and stochastic models: The corresponding state lifetimes in the deterministic model are equal to the exponential mean of the lifetimes in the stochastic model. An important implication is therefore that the demographics of an exponential culture will be well-fit by a deterministic model even if the state timing is stochastic. Although we explore the implications of the models in the context of the Escherichia coli cell cycle, we expect both the models as well as the significance of the exponential-mean lifetimes to find many applications in the quantitative analysis of cell cycle dynamics in other biological systems.

biophysics↗

bioRxiv2020

Replication-transcription conflicts promote adaptive evolution

Cells may be able to promote adaptive evolution in a gene-specific and temporally-controlled manner. Genes encoded on the lagging strand have a higher mutation rate and evolve faster than genes on the leading strand. This effect is likely driven by head-on replication-transcription conflicts, which occur when lagging strand genes are transcribed during DNA replication. We previously suggested that the ability to selectively increase mutagenesis in a subset of genes may provide an adaptive advantage for cells. However, it is also possible that this effect could be neutral or even highly deleterious. Distinguishing between these models is important because, if the adaptive model is correct, it would indicate that 1) head-on conflicts, which are generally deleterious, can also provide a benefit to cells, and 2) cells possess the remarkable ability to fine-tune adaptive evolution. Furthermore, investigating these models may address the long-standing debate regarding whether accelerated evolution through conflicts can be adaptive. To distinguish between the adaptive and neutral models, we conducted single nucleotide polymorphism (SNP) analyses on wild strains of bacteria, from divergent phyla. To test the adaptive hypothesis, we analyzed convergent mutation patterns. As a simple test of the neutral hypothesis, we performed in silico modeling. Our results show that convergent mutations are enriched in lagging strand genes and that these mutations are unlikely to have arisen by chance. Additionally, we observe that convergent mutation frequency has a stronger positive correlation with gene-length in lagging strand genes. This effect strongly suggests that head-on conflicts between the DNA replication and transcription machineries are a key mechanism driving the formation of convergent mutations. Together, our data indicate that head-on replication-transcription conflicts can promote adaptive evolution in a variety of bacterial species, and potentially other organisms.

evolutionary biology↗

bioRxiv2020

Genome-wide association and function studies identify Mfd as a critical RNA polymerase co-factor at hard-to-transcribe regions

RNA polymerase (RNAP) encounters various roadblocks during transcription. These obstacles can impede RNAP movement, influence transcription, ultimately necessitating the activity of RNAP associated factors. One such factor is the bacterial protein Mfd; a highly conserved DNA translocase and evolvability factor that interacts with RNAP. Although Mfd is thought to function primarily in the repair of DNA lesions that stall RNAP, increasing evidence suggests that it may also be important for transcription regulation. However, this is yet to be fully characterized. To shed light on Mfds in vivo functions, we identified the chromosomal regions where it associates. We analyzed Mfds impact on RNAP association and transcription regulation genome-wide. We found that Mfd represses RNAP association at many chromosomal regions. We found that these regions show increased RNAP pausing, suggesting that they are hard-to-transcribe. Interestingly, we noticed that the majority of the regions where Mfd regulates transcription contain highly structured regulatory RNAs. The RNAs identified regulate a myriad of biological processes, ranging from metabolism, to tRNA regulation, to toxin-antitoxin (TA) functions. We found that transcription regulation by Mfd, at least at some TA loci, is critical for cell survival. Lastly, we found that Mfd promotes mutagenesis in at least one toxin gene, suggesting that its function in regulating transcription may promote evolution of certain TA systems, and other regions containing strong RNA secondary structures. We conclude that Mfd is an RNAP co-factor that is important, and at times critical, for transcription regulation at hard-to-transcribe regions, especially those that express structured regulatory RNAs. SignificanceThe bacterial DNA translocase Mfd binds to stalled RNAPs and is generally thought to facilitate transcription-coupled DNA repair. Most of our knowledge about Mfd is based on data from biochemical studies. However, little is known about Mfds function in living cells, especially in the absence of exogenous DNA damage. Here, we show that Mfd modulates RNAP association and alters transcription at a variety of chromosomal loci, especially those containing highly structured, regulatory RNAs. As such, this work improves our understanding of Mfds function in living cells, and assigns it a new function as a transcription regulator.

molecular biology↗

bioRxiv2020

The recent report by Liu and Zhang Claiming "No support for the adaptive hypothesis of lagging-strand encoding in bacterial genomes" is factually incorrect

Several previous studies by our group suggest that positive selection can drive certain (not all) genes to be retained in the lagging strand orientation2,5,6. This is likely the result of multiple factors including accelerated evolution through replication-transcription conflicts. Liu and Zhang challenge this view, and claim that our method for detecting gene inversions is flawed. Below, we provide empirical evidence that their criticisms are largely unfounded, and show that our original analysis and conclusions are well supported. Though the GC skew method does have a detection limit, we provide new evidence that the fundamental assumptions of our model are accurate, and introduce an improved GC skew calculation which correctly identified 100% of the authors gene inversions. Our new findings indicate that the trends we originally identified are stronger than they initially appeared: across species, 89-96% of lagging strand genes appear to be natively leading strand genes that changed orientation. Our statistical analyses offer further support for the notion that for some genes, the lagging strand orientation can be adaptive.

evolutionary biology↗

bioRxiv2019

Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts

Conflicts between the replication and transcription machineries have profound effects on chromosome duplication, genome organization, as well as evolution across species. Head-on conflicts (lagging strand genes) are significantly more detrimental than co-directional conflicts (leading strand genes). The source of this fundamental difference is unknown. Here, we report that topological stress underlies this difference. We find that head-on conflict resolution requires the relaxation of positive supercoils DNA gyrase and Topo IV. Interestingly, we find that after positive supercoil resolution, gyrase introduces excessive negative supercoils at head-on conflict regions, driving pervasive R-loop formation. The formation of these R-Loops through gyrase activity is most likely caused by the diffusion of negative supercoils through RNA polymerase spinning. Altogether, our results address a longstanding question regarding replication-transcription conflicts by revealing the fundamental mechanistic difference between the two types of encounters.

molecular biology↗

bioRxiv2018

Gene inversion increases evolvability in bacteria

In bacteria, most genes are encoded on the leading strand, co-orienting the movement of the replication machinery with RNA polymerases. This co-orientation bias reduces the frequency of highly detrimental head-on collisions between the two machineries. This and other work set up the expectation that over evolutionary time, head-on alleles are selected against, maximizing genome co-orientation. Our findings challenge this model. Using the well-established GC skew method, we reveal the evolutionary inversion record of all chromosomally encoded genes in multiple divergent bacterial pathogens. We find that a surprisingly large number of co-oriented genes have inverted to, and are retained in the head-on orientation. Furthermore, we find that these head-on genes, (including key antibiotic resistance and virulence genes) have higher rates of nonsynonymous mutations and are more frequently under positive selection (dN/dS>1). Based on these results, we propose that bacteria increase their evolvability through gene inversion and promotion of head-on replication-transcription collisions.

evolutionary biology↗

bioRxiv2017

Head-on replication-transcription collisions lead to formation of life threatening R-loops

Encounters between transcription and DNA replication machineries lead to conflicts that shape genomes, influence evolution, and lead to genetic diseases in humans. Although unclear why, head-on transcription (lagging strand genes) is especially disruptive to replication, increases DNA breaks, and promotes mutagenesis. Here, we show that head-on replication-transcription conflicts lead to pervasive RNA:DNA hybrid formation in Bacillus subtilis. We find that replication beyond head-on conflict regions requires the activity of a RNA:DNA hybrid processing enzyme, RNase HIII. Remarkably, pervasive RNA:DNA hybrid formation at head-on genes completely stops replication and inhibits gene expression in a replication-dependent manner. Accordingly, we find that resolution of head-on conflicts by RNase HIII is crucial for survival upon exposure to various stresses, as many stress response genes are encoded head-on to replication. We conclude that R-loops, RNA:DNA hybrids formed outside of the transcription bubble, exacerbate head-on replication-transcription conflicts, thereby threatening life, especially upon exposure to environmental stresses.

molecular biology↗

bioRxiv2016

The replisomes remain spatially proximal throughout the cell cycle in bacteria

The positioning of the DNA replication machinery (replisome) has been the subject of several studies. Two conflicting models for replisome localization have been proposed: In the Factory Model, sister replisomes remain spatially colocalized as the replicating DNA is translocated through a stationary replication factory. In the Track Model, sister replisomes translocate independently along a stationary DNA track and the replisomes are spatially separated for the majority of the cell cycle. Here, we used time-lapse imaging to observe and quantify the position of fluorescently labeled processivity-clamp (DnaN) complexes throughout the cell cycle in two highly-divergent bacterial model organisms: Bacillus subtilis and Escherichia coli. Because DnaN is a core component of the replication machinery, its localization patterns should be an appropriate proxy for replisome positioning in general. We present automated statistical analysis of DnaN positioning in large populations, which is essential due to the high degree of cell-to-cell variation. We find that both bacteria show remarkably similar DnaN positioning, where any potential separation of the two replication forks remains below the diffraction limit throughout the majority of the replication cycle. Additionally, the localization pattern of several other core replisome components is consistent with that of DnaN. These data altogether indicate that the two replication forks remain spatially colocalized and mostly function in close proximity throughout the replication cycle.The conservation of the observed localization patterns in these highly divergent species suggests that the subcellular positioning of the replisome is a functionally critical feature of DNA replication.\n\nAuthor SummaryCell proliferation depends on efficient replication of the genome. Bacteria typically have a single origin of replication on a circular chromosome. After replication initiation, two replisomes assemble at the origin and each copy one of the two arms of the chromosome until they reach the terminus. There have been conflicting reports about the subcellular positioning and putative co-localization of the two replication forks during this process. It has remained controversial whether the two replisomes remain relatively close to each other with the DNA being pulled through, or separate as they translocate along the DNA like a track. Existing studies have relied heavily on snapshot images and these experiments cannot unambiguously distinguish between these two models: i.e. two resolvable forks versus two pairs of co-localized forks. The ability of replication to re-initiate before cell division in bacterial cells further complicates the interpretation of these types of imaging studies. In this paper, we use a combination of snapshot imaging, time-lapse imaging, and quantitative analysis to measure the fraction of time forks are co-localized during each cell cycle. We find that the forks are co-localized for the majority ( 80%) of the replication cycle in two highly-divergent model organisms: B. subtilis and E. coli. Our observations are consistent with proximal localization of the two forks, but also some transient separations of sister forks during replication. The conserved behavior of sub-cellular positioning of the replisomes in these two highly divergent species implies a potential functional relevance of this feature.

cell biology↗